Following obtaining support from the Director, NCI, Dr. Waldmann directed an inter-institute collaboration (NCI-NIAID) directed toward the production under current good manufacturing practices (cGMP) of IL-15 for clinical trials in patients with metastatic malignant melanoma and renal cell cancer as well as in patients with HIV/AIDS receiving HAART therapy. In a modification of an expression system and purification method developed in the Waldmann Laboratory, the Biopharmaceutical Development Program (BDP) of the National Cancer Institute has produced recombinant human IL-15 (rhIL-15) under current good manufacturing practices (cGMP). In this work, an E. coli based fermentation and purification process was developed for the production of clinical grade recombinant human IL-15. DNA sequences from human IL-15 were inserted into pET-28b plasmid, were expressed in the E. coli host BL21-AL. The inclusion bodies of IL-15 produced in E. coli were solubilized, refolded and orthogonally purified to yield active IL-15. RhIL-15 was produced as a non-glyclosylated, single-chain polypeptide of 115 amino acids, having a calculated molecular weight of 12,901 Da. Purified rhIL-15 showed in vitro activity in a CTLL-2 cell proliferation assay in the range of 15-25 x 106 IU/mg protein standardized against the international standard obtained from the National Institute of Biological Standard and Control (NIBSC). In a toxicology study directed by the Waldmann Laboratory, the rhIL-15 was evaluated for safety (toxicity), pharmacokinetics, immunogenicity, autoimmunity and impact on elements of the normal immune system of rhIL-15 in rhesus macaques. The only laboratory abnormally was a grade 3/4 neutropenia that was secondary to a redistribution of neutrophils from the circulation to the tissues. No abnormalities were observed following IL-15 administration in gross or microscopic analysis of tissues at autopsy. The animals did not produce antibodies to IL-15 as assessed using a sensitive 2-armed capture technique developed in the Waldmann Laboratory. IL-15 administration was associated with significant increases in the number of circulating NK and CD3 CD8 especially central and effector memory T-lymphocytes. The administration was also associated with a tenfold increase in the circulating IL-2R alpha levels. Under the supervision of Dr. Waldmann rhIL-15 has been produced under cGMP conditions. Request for approval has been initiated for a clinical trial Phase I Study of Intravenous Recombinant Human IL-15 (rhIL-15) in Adults with Refractory Metastatic Malignant Melanoma and Metastatic Renal Cell Cancer. The sponsor is the Metabolism Branch, CCR, NCI with Dr. Thomas A. Waldmann as Study Chair and IND holder. The study will be performed on the Metabolism Branch, CCR, NCI in the Clinical Center with Dr. John C. Morris as the principal investigator (PI). The primary objective of this trial is to determine the safety, toxicity profile, dose-limiting toxicity and maximum tolerated dose of intravenous recombinant human IL-15 produced by the BDP and administered as a daily intravenous infusion for 12 days in individuals with incurable metastatic malignant melanoma and renal cell cancer. The secondary objectives include determination of rhIL-15 pharmacokinetics and to determine the immunogenicity of the IL-15 in individuals receiving this drug. Phenotypic analysis of circulating lymphocytes will be used to define the biological effects of rhIL-15. A final objective is to obtain preliminary information on the efficacy of IL-15 in the treatment of patients with metastatic malignancy. Following the completion of the phase I trial in patients with metastatic malignancy, phase I/II trials will be initiated in collaboration with Dr. Clifford Lane of NIAID for the evaluation of rhIL-15 in the treatment of patients with HIV/AIDS receiving HAART therapy.